阅读说明：本技术 一种外泌体水凝胶伤口敷料及其制备方法 (Exosome hydrogel wound dressing and preparation method thereof ) 是由 王立生 聂文博 赵丽晶 吴祖泽 于 2020-03-26 设计创作，主要内容包括：本发明属于生物医学技术领域,具体涉及一种外泌体水凝胶伤口敷料及其制备方法。本发明外泌体水凝胶伤口敷料,包括间充质干细胞外泌体和水凝胶,其中水凝胶由普朗尼克F127和普朗尼克F68组成。分别配制普朗尼克F127和普朗尼克F68的生理盐水溶液,及间充质干细胞外泌体生理盐水重悬液,在4℃条件下将上述溶液混合,即可得到外泌体水凝胶伤口敷料。实验结果表明,相对于单独采用间充质干细胞外泌体和水凝胶,本发明制备得到的伤口敷料更能促进皮肤创面细胞再生,缩短伤口愈合时间。(The invention belongs to the technical field of biomedicine, and particularly relates to an exosome hydrogel wound dressing and a preparation method thereof. The exosome hydrogel wound dressing comprises mesenchymal stem cell exosomes and hydrogel, wherein the hydrogel is composed of pluronic F127 and pluronic F68. Respectively preparing physiological saline solution of pluronic F127 and pluronic F68 and mesenchymal stem cell exosome physiological saline resuspension, and mixing the solutions at 4 ℃ to obtain the exosome hydrogel wound dressing. The experimental result shows that compared with the method of singly adopting the mesenchymal stem cell exosome and the hydrogel, the wound dressing prepared by the invention can better promote the regeneration of skin wound cells and shorten the wound healing time.)

1. An exosome hydrogel wound dressing characterized by: the dressing comprises mesenchymal stem cell exosomes and a hydrogel consisting of pluronic F127 and pluronic F68.

2. The wound dressing of claim 1, wherein the concentration of pluronic F127 in the wound dressing is 200-220mg/m L and the concentration of pluronic F68 in the wound dressing is 25-100mg/m L.

3. The wound dressing of claim 1, wherein the concentration of the mesenchymal stem cell exosomes in the wound dressing is 100-200 μ g/m L.

4. A wound dressing according to any one of claims 1 to 3, wherein: the volume ratio of the mesenchymal stem cell exosomes to the hydrogel is 1: 1.

5. A method of preparing an exosome hydrogel wound dressing according to any one of claims 1 to 4, characterised in that a solution of pluronic F127 and pluronic F68 in physiological saline is prepared to obtain solution A; preparing a mesenchymal stem cell exosome physiological saline resuspension to obtain a solution B; and mixing the solution A and the solution B at 4 ℃ to obtain the exosome hydrogel wound dressing.

6. The method as claimed in claim 5, wherein the concentration of Pluronic F127 in said solution A is 400-440mg/m L, and the concentration of Pluronic F68 is 50-200mg/m L.

7. The method for preparing the mesenchymal stem cell exosome according to claim 5, wherein the concentration of the mesenchymal stem cell exosome in the liquid B is 200-400 μ g/m L.

8. The production method according to claim 6 or 7, wherein the solution A and the solution B are mixed in a volume ratio of 1: 1.

9. The method of claim 5, wherein the mesenchymal stem cell exosomes are extracted from a supernatant of a mesenchymal stem cell culture medium.

10. The preparation method according to claim 9, wherein the extraction method comprises the steps of transferring the supernatant into a 50m L centrifuge tube, centrifuging at the temperature of 4 ℃ for 5min at the speed of 500Xg, collecting the supernatant, transferring into a new 50m L centrifuge tube, centrifuging at the temperature of 4 ℃ for 30min at the speed of 2000Xg, collecting the supernatant, mixing with precooled 16% PEG6000 in the same volume, fully inverting and mixing, standing overnight at the temperature of 4 ℃, centrifuging at the temperature of 4 ℃ for 60min at the speed of 10000Xg, pouring out the supernatant, adding 50-100 mu L precooled sterile PBS for resuspension to obtain the crude mesenchymal dry cell exosomes, adding 15-20m L precooled sterile PBS for uniformly mixing, centrifuging at the temperature of 4 ℃ for 100000Xg, centrifuging for 70min, discarding the supernatant, and adding 200-400 mu L sterile PBS to obtain the mesenchymal dry cell exosomes.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to an exosome hydrogel wound dressing and a preparation method thereof.

Background

Statistically, more than 2% of our country experience chronic wound healing, with costs as high as 150 billion dollars per year worldwide for wound care products. Traditional wound dressings such as chitosan, hyaluronic acid, alginate, gelatin and the like have certain adjuvant therapy effect on chronic wounds, but the healing degree of the wounds still mainly depends on the self-repairing capability of patients, so that a large number of chronic refractory wounds are not healed for a long time and even die due to poor nutritional state, advanced age, low immunity and other reasons of the patients. Therefore, there is an urgent need to develop a novel dressing with tissue regeneration function based on clinical wound care needs.

The mesenchymal stem cell exosome (MSC-exosome) has the functions similar to those of a source cell, has high efficiency on the action of a target cell, is beneficial to long-term stable storage and transportation, is easy to control the use concentration and dosage, and can overcome the hidden troubles of low survival rate of the mesenchymal stem cell in vivo and easy mutation and tumorigenesis in long-term use. The wound dressing usually takes hydrogel as a bracket to carry effective components, so as to achieve the effect of healing the wound. However, the traditional hydrogel such as chitosan and silver ion dressing has good sterilization and bacteriostasis effects, but has certain toxicity, certain damage to skin and soft tissue and influence on wound healing. In addition, the hydrogel of the wound dressing is generally a colloidal solid substance, is prepared by complex and high-cost biomolecules through complex reactions, generally needs to be crosslinked, has a complex preparation method, and easily has the defects that the hydrogel and active ingredients are not uniformly mixed, the slow release effect cannot be achieved, and the effect of the active ingredients is influenced, so the dressing prepared by the existing hydrogel and the endocrine system still has the defect of short healing time.

Disclosure of Invention

The invention provides an exosome hydrogel wound dressing and a preparation method thereof, which can obviously promote wound surface cell regeneration and shorten wound healing time.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention provides an exosome hydrogel wound dressing which comprises mesenchymal stem cell exosomes and hydrogel, wherein the hydrogel comprises pluronic F127 and pluronic F68.

Preferably, the concentration of pluronic F127 in the wound dressing is 200-220mg/m L and the concentration of pluronic F68 in the wound dressing is 25-100mg/m L.

Preferably, the concentration of mesenchymal stem cell exosomes in the wound dressing is 100-200 μ g/m L.

Preferably, the volume ratio of the mesenchymal stem cell exosomes to the hydrogel is 1: 1.

The invention also provides a preparation method of the exosome hydrogel wound dressing, which comprises the following steps: preparing physiological saline solution of pluronic F127 and pluronic F68 to obtain solution A; preparing a mesenchymal stem cell exosome physiological saline resuspension to obtain a solution B; and mixing the solution A and the solution B at 4 ℃ to obtain the exosome hydrogel wound dressing.

Preferably, in the solution A, the concentration of the pluronic F127 is 400-440mg/m L, and the concentration of the pluronic F68 is 50-200mg/m L.

Preferably, in the liquid B, the concentration of the mesenchymal stem cell exosomes is 200-400 mu g/m L.

Preferably, the liquid A and the liquid B are mixed according to the volume ratio of 1: 1.

Preferably, the mesenchymal stem cell exosome is extracted from a supernatant of a mesenchymal stem cell culture medium.

Preferably, the supernatant is kept at PEG 60004 ℃ overnight, and the precipitate obtained after centrifugation is resuspended in PBS to obtain the mesenchymal stem cell exosome.

Compared with the prior art, the invention has the following beneficial effects:

the exosome hydrogel wound dressing comprises mesenchymal stem cell exosomes and hydrogel, wherein the hydrogel is composed of pluronic F127 and pluronic F68. Wherein the mesenchymal stem cell exosome can promote the secretion of collagen and elastin activated by fibroblasts, induce epithelial cell proliferation and angiogenesis, promote re-epithelialization of skin wounds and prevent myofibril formation; the hydrogel prepared from pluronic F127 and pluronic F68 can provide a moist microenvironment for skin wounds, absorb wound exudates, protect the wounds from infection, and accelerate wound healing. The hydrogel is used as a medicine carrying system and carries the mesenchymal stem cell exosomes, so that the local action time of the mesenchymal stem cell exosomes can be prolonged, and the effect of the hydrogel on promoting wound healing is enhanced. The experimental result shows that compared with the independent adoption of the mesenchymal stem cell exosome and the hydrogel, the wound dressing disclosed by the invention can better promote the regeneration of skin wound cells and shorten the wound healing time.

The hydrogel adopted by the invention is used as a composite tissue engineering scaffold material for bearing the mesenchymal stem cell exosomes. The prepared exosome hydrogel wound dressing is in a liquid state at low temperature, is in a gel state when being externally applied to skin when being used, can improve the contact area between the dressing and the wound, promotes the healing of the skin wound, and is a novel new material for wound care. The hydrogel disclosed by the invention does not need complex high-cost biomolecules, is simple in preparation method, can cause complex cellular reaction through simple chemical functional groups, and is a cost-saving and non-toxic scaffold material.

Drawings

FIG. 1 is a graph of the effect of different concentrations of Pluronic F127 on the survival of C2C12 cells;

FIG. 2 is a graph of the effect of different concentrations of pluronic F68 on C2C12 cell survival;

FIG. 3 shows the skin wound status of mice in each group at each time point;

fig. 4 is the area of the wound surface at different time points after treatment of skin wounds for each group.

Detailed Description

The invention provides an exosome hydrogel wound dressing which comprises mesenchymal stem cell exosomes and hydrogel, wherein the hydrogel is composed of pluronic F127 and pluronic F68.

In the invention, the concentration of the mesenchymal stem cell exosome in the wound dressing composition is 100-200 mu g/m L. the mesenchymal stem cell exosome has the function of promoting the regeneration of damaged histiocytes similar to the source cells, and is more beneficial to promoting the repair of skin wound surfaces.

In the present invention, the concentration of pluronic F127 in the wound dressing composition is 200-220mg/m L and the concentration of pluronic F68 in the wound dressing composition is 25-100mg/m L.

The pluronic is temperature sensitive hydrogel which is liquid at low temperature and solid at higher temperature, and the pluronic with different concentrations has different phase transition temperatures. If the phase transition temperature of the temperature-sensitive hydrogel is too high, the hydrogel is solidified in advance, cannot be fully mixed with exosomes at the later stage, cannot be fully contacted with a wound surface, and the treatment effect is influenced; if the phase transition temperature is too low, the gel can not be formed after being smeared on a wound, is easy to run off in a liquid state, and cannot play a role in sustained-release treatment. By measuring the phase transition temperatures of mixtures of different concentrations of pluronic F127 and pluronic F68, as shown in table 1 below:

TABLE 1 mixture of different concentrations of Pluronic F127 and Pluronic F68 and phase transition temperature

As can be seen from Table 1, the phase transition temperature of the gel B3 is 35.1 ℃, the phase transition temperature of the gel C1 is 36.1 ℃, and the temperature is closest to the temperature of a wound, so that the exosome hydrogel wound dressing can be fully mixed with exosomes in the preparation process, can be solidified on the wound in the use process, and plays a role in slow-release treatment.

The invention also provides a preparation method of the exosome hydrogel wound dressing, which comprises the following steps of preparing a normal saline solution with the concentration of 400-440mg/m L pluronic F127 and the concentration of 50-200mg/m L pluronic F68 to obtain a solution A, preparing a mesenchymal stem cell exosome normal saline resuspension with the concentration of 200-400 mu g/m L to obtain a solution B, and mixing the solution A and the solution B at the temperature of 4 ℃ to obtain the exosome hydrogel wound dressing.

At present, the hydrogel for the wound dressing is generally a solid substance, generally needs to be subjected to a crosslinking reaction, has a complex preparation method, and easily has the defects that the hydrogel and active ingredients are not uniformly mixed, the slow release effect cannot be achieved, and the effect of the active ingredients is influenced. The hydrogel adopted in the invention is temperature-sensitive hydrogel which is in a liquid state at low temperature, and is easy to be uniformly mixed with exosome in the preparation process, the preparation method is simple, the preparation process is carried out in a low-temperature environment, and the effect of exosome can be effectively prevented from being damaged. In the process of treating the wound, the dressing of the invention not only does not need to be frequently replaced, but also can achieve the effects of slow release and wound healing promotion. In addition, the contact area of the dressing of the invention and the wound is larger, the dressing is easier to absorb the exudate of the soft tissue, and the wound healing time is shortened.

In the invention, the concentration of the mesenchymal stem cell exosome in the liquid B is 200-400 mu g/m L.

In the invention, the liquid A and the liquid B are mixed according to the volume ratio of 1: 1.

The preparation method is characterized in that the mesenchymal stem cell exosome is extracted from supernatant of a mesenchymal stem cell culture medium, the supernatant is transferred to a 50m L centrifuge tube and centrifuged at 4 ℃, the supernatant is transferred to a new 50m L centrifuge tube and centrifuged at 4 ℃ at 2000Xg for 30min and is collected and mixed with precooled 16% PEG6000 isopyknic PBS, the mixture is fully inverted and mixed uniformly, the mixture is kept stand in a 4 ℃ environment, the mixture is centrifuged at 4 ℃ at 10000Xg and 60min, the supernatant is poured out and is added with 50-100 mu of L sterile PBS, namely, crude mesenchymal stem cell exosome is resuspended, the crude mesenchymal stem cell exosome is added with 100020-20 mu of sterile dry cell exosome, the supernatant is added with 100020-36 mu of sterile dry cell exosome and is mixed uniformly, the supernatant is added with precooled at 100020-400 mu of dry cell exosome, the supernatant is added with precooled dry cell exosome, and is added with 10002-100000 mu of sterile dry cell exosome, and is added with precooled and is added with sterile dry cell exosome and is added with sterile dry cell resuspension at 20-400 mu of sterile dry cell supernatant, and sterile dry cell resuspension is added with sterile dry cell resuspension temperature.

The hydrogel is used as a medicine carrying system and carries the mesenchymal stem cell exosomes, so that the local action time of the mesenchymal stem cell exosomes can be prolonged, and the effect of promoting wound healing of the hydrogel is enhanced. Compared with the method of singly adopting the mesenchymal stem cell exosome and the hydrogel, the wound dressing disclosed by the invention can be used for better promoting the regeneration of skin wound cells and shortening the wound healing time. The exosome hydrogel wound dressing prepared by the invention is in a liquid state at low temperature, is in a gel state when being externally applied to skin during use, can improve the contact area between the dressing and the wound, promotes the healing of the skin wound, and is a novel new material for wound care. The hydrogel disclosed by the invention does not need complex high-cost biomolecules, is simple in preparation method, can cause complex cellular reaction through simple chemical functional groups, and is a cost-saving and non-toxic scaffold material.

The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.